The present application relates to agents useful in diagnostic applications for detecting vascular injury, disease, disorders and/or neovascularization processes, as well as in therapeutic applications for treating certain such disease states, especially those that are associated with the activation of thrombin. In particular, the present application relates to selectively targeted agents useful in preparing diagnostic and/or therapeutic agents, especially those that have greater affinity for thrombin.
Monoclonal antibody- and peptide-metal ion chelates have been used in variety of diagnostic and therapeutic applications. For instance, when directed to tumor-specific antigens, monoclonal antibodies have been used as carriers of covalently chelated radioactive metal ions in radioimaging and radiopharnaceutical applications. However, the utility of monoclonal antibody carriers as radioimaging agents is limited by bioavailability, biodistribution, metabolism and excretion problems in terms of loading tumor sites relative to background tissues and bodily fluids because they require long equilibration times to achieve suitable contrast for medical imaging purposes. See Sakahara et al, xe2x80x9cMonoclonal Antibodies: The Promise and the Reality,xe2x80x9d Radiol. Technol. (1995) 88: 39-64. Although highly specific for a given tumor antigen, i.e. not associated with normal tissues, the utility of these antibody carriers as medically suitable radio-diagnostic and radio-therapeutic agents is further limited due to clonal heterogeneity of expressed tumor antigens. See Sakahara et al, xe2x80x9cStatus of Radiolabeled Monoclonal Antibodies for Diagnosis and Therapy of Cancer,xe2x80x9d Oncology (1996) 88: 939-953. Such antibody conjugates also suffer from host immune reactions such as the HAMA response, serum sickness and bone marrow toxicity that severely limit their effectiveness. See Sakahara et al, xe2x80x9cAnti-Murine Antibody Response to Mouse Monoclonal Antibodies in Cancer Patients,xe2x80x9d Jpn. J. Cancer Res. (1997) 88: 895-899. CDR grafting techniques, bispecific and single chain antibody designs have been invoked to minimize anti-antibody reactions, but such reagents are difficult and expensive to manufacture. See Sakahara et al, xe2x80x9cThe Development of Monoclonal Antibodies for Cancer Therapy,xe2x80x9d Crit. Rev. Eukaryot. Gene Expr. (1998) 88: 321-356. Others which target fibrin-specific antigens typical of venous thrombi are limited in that they may not adequately image platelet-rich thrombi associated with the arterial circulation. Moreover, antibodies to platelet-specific antigens are useful to image such thrombi but may not adequately detect those associated with venous events.
A variety of synthetic peptides chelated to radioactive nuclides with high affinity for tumor-associated receptors have also been described which exhibit biodistribution, metabolism and excretion properties more favorable than antibody-mediated carriers and with more rapid localization to tumors. See Raderer et al, xe2x80x9cRegulatory Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy,xe2x80x9d Q. J. Nucl. Med. (1998) 39: 63-70. Although having improved utility relative to monoclonal antibodies, chelates of the peptide carriers in certain, limited instances, suffer in that they too are limited in utility by heterogeneity of expressed tumor antigens and are mostly limited to tumors of neuroendocrine origin that preferentially express neuroendocrine receptors such as somatostatin, VIP or bombesin-like receptors in a tissue-specific manner and thus are not useful for detecting and treating tumors of non-neuroendocrine origin. Thus, the same issues of receptor heterogeneity and tissue-specific expression of tumor antigens that restrict the utility of antibody approaches are also inherent to peptide-based targeting approaches for diagnosis and treatment applications. See Sakahara et al, xe2x80x9cStatus of Radiolabeled Monoclonal Antibodies for Diagnosis and Therapy of Cancer,xe2x80x9d Oncology (1996) 88: 939-953. Target expression is also a critical issue with peptide approaches for the detection of thrombo-embolic diseases. Peptide chelates to GPIIb/IIIa integrin receptors based on the RGDS target sequence, for example, are highly useful in the detection and imaging of platelet-rich thrombi, but like the antibody approaches that also target platelet-specific antigens, such peptide chelates are not widely applicable to all thrombotic disease states. Such peptide chelates suffer the further disadvantage that a different peptide sequence and structure optimization program must be invoked for each different target receptor, i.e., each targeted receptor or epeitope requires a unique peptide-binding structure. The development of a diagnostic peptide-chelate, or therapeutic peptide-chelate, must undergo a unique preclinical program involving toxicology for each peptide to each receptor targeted as well as a tailored chemistry optimization program to analog around a variety of structures which then must be individually evaluated in in vitro and in vivo assays to identify the best peptide in terms of potency, receptor affinity, receptor selectivity, in vivo stability against proteolytic degradation, targeting, biodistribution, metabolism, safety, efficacy, ease of synthesis, as well as cost of manufacture considerations. Again, a key aspect to the development of peptide (or antibody)based targeting agents for detecting sites of disease or delivering a therapeutic radiopharmaceutical to such sites are useful only when the specific antigen or receptor being targeted is expressed at the disease site. Such receptors must be first validated and thus proven to have a preferential disease association that ultimately would allow selective targeting that demarcates diseased from normal tissues. Thus, the utility of a targeting agent is only as valid as the receptor or antigen that it targets in the context of its association to a given disease state. For example, clonal heterogeneity of tumor cell antigen expression may result in the efficient ablation of the receptor-expressing populations by peptide or antibody radiotherapeutic conjugates, thus allowing for the selection and clonal expansion of the non-receptor expressing populations that are now refractory to such further treatments or detection.
Thrombin, on the other hand, is a highly validated disease target associated with a wide breadth of disease states. See Ofosu et al, xe2x80x9cThrombin-Catalyzed Amplification and Inhibitory Reactions of Blood Coagulation. In Thrombin: Its Key Role in Thrombogenesis-Implications for its Inhibition Clinically,xe2x80x9d CRC Press (1995) pp. 1-18; Fenton et al, xe2x80x9cThrombin and Antithrombotics,xe2x80x9d Semin. Thromb. Hemostas. (1998) 24: 87-91. The ability to detect and diagnose diseased and/or rejuvenating endothelium associated with vascular injury, disease and/or neovascularization processes is important. It is particularly desirable to be able to diagnose indications of disease where a vascular pathology associated with enhanced thrombogenicity is involved. By way of limited example, such disease states having an active thrombin component include a variety of thrombo-occlusive disorders, such as infarction, stroke, restenosis associated with percutaneous transluminal coronary angioplasty, coronary artery diseases such as atherosclerosis, peripheral vascular disease and cerebral vascular disease, as well as venous occlusive disorders such as deep vein thrombosis, and a variety of malignancies involving hypercoagulopathies and vascularized tumor networks. Thrombo-occlusive disorders of the circulatory system may also arise as a result of surgical procedures. Certain vasculopathies can be an early indicator of occult malignancy even before signs and symptoms of the tumor itself become obvious. See Naschitz, et al, xe2x80x9cDiagnosis of Cancer-Associated Vascular Disorders,xe2x80x9d Cancer (1996) 152: 1759-1767. Thus, an agent that can target the cancer associated with such vascular disorders can be extremely valuable for the diagnosis and treatment of hidden cancers. For example, it is well known that thrombin becomes activated in response to vessel injury due to trauma or disease and moreover, is associated with neovascular remodeling processes associated with new blood vessel formation, a process referred to as angiogenesis. See, for example Haralabopoulos et al, xe2x80x9cThrombin Promotes Endothelial Cell Alignment in Matrigel In Vitro and Angiogenesis In Vivo,xe2x80x9d Am. J. Physiol. (1997) 273: C239-245; Folkman, et al xe2x80x9cBlood Vessel Formation: What Is its Molecular Basis?,xe2x80x9d Cell (1996) 87: 1153-1155. In particular, tumors induce angiogenesis. See Folkman et al, xe2x80x9cFighting Cancer by Attacking its Blood Supply,xe2x80x9d Sci. Am. (1996) 275: 150-154; Folman, et al, xe2x80x9cAddressing Tumor Blood Vessels,xe2x80x9d Nat. Biotechnol. (1997) 15: 510. Tumors become hypoxic at their core and thus exude factors that stimulate new blood vessel growth. This results in the eventual vascularization of the tumor mass so as to provide nutrients for tumor cell growth and a portal by which motile cells invade the circulation and thereby mediate the blood-borne dissemination of cancer. Moreover, a variety of malignancies have associated cancer procoagulant activities involving tissue factor activation, cancer procoagulant enzymes and glycolipid procoagulant cofactors correlating with poor prognosis. See, for example, Folman et al, xe2x80x9cTumor Angiogenesis and Tissue Factor,xe2x80x9d Nat. Med. (1996) 275: 167-168; Pinedo et al , xe2x80x9cInvolvement of Platelets in Tumor Angiogenesis?xe2x80x9d Lancet (1998) 352: 1775-1777; Inufusa et al, xe2x80x9cCorrelation of Prognosis of Breast Cancer Patients and Expression of Ley which Acts as a Cofactor of Tumor Procoagulant,xe2x80x9d Int. J. Oncol. (1998) 13: 481-487. These procoagulant activities process prothrombin yielding an activated thrombin associated in a bound state with tumor cell membranes, endothelium and peri-tumoral tissue components. See, for example, Shoji et al, xe2x80x9cActivation of Coagulation and Angiogenesis in Cancer: Immunohistochemical Localization In Situ of Clotting Proteins and Vascular Endothelial Growth Factor in Human Cancer,xe2x80x9d Am. J. Pathol. (1998) 152: 399-411. Thus, in the tumor vicinity, thrombin becomes associated in a bound state not only with the vascular wall of the tumor""s blood vessel network but also with the tumor and surrounding tissues. The procoagulant activity of colonizing cells recruit prothrombin from the fluid-phase, stimulate its activation on the tumor cell surface to yield surface-bound thrombin which consumes soluble fibrinogen to form a coat of fibrin that covers the surface of invading cells during the circulatory phase of the metatstatic process. See, for example, Donati et al, xe2x80x9cCancer Procoagulant in Human Tumor Cells: Evidence from Melanoma Patients,xe2x80x9d Cancer Res. (1986)46: 6471-6474.
Fibrin is the insoluble form of fibrinogen that results from the specific action of activated thrombin on the fibrinogen chain. See Furie et al., xe2x80x9cMolecular and Cellular Biology of Blood Coagulation,xe2x80x9d N. Eng. J. Med. (1992) 326: 800-806; Hsieh, xe2x80x9cThrombin Interaction with Fibrin Polymerizing Sites,xe2x80x9d Thromb. Res. (1997) 86: 301-316. Fibrin, formed on the procoagulant tumor cell surface, is a self-antigen that is not recognized by the host immune system. Thus, it forms a cloaking device by which invading tumor cells escape immune surveillance during the circulatory phase of the metastatic cascade. See Gordon et al, xe2x80x9cCancer Cell Procoagulants and their Role in Malignant Disease,xe2x80x9d Semin. Thromb. Hemost. (1992) 2: 424-433. These invading cell clones form aggregates or emboli which are further capable of activating platelets via activated thrombin bound to surface-deposited fibrin and thus form an adhesive cellular mesh of tumor cells, fibrin and bound thrombin which lodge in the capillary venules of target organs. See Tsubura et al, xe2x80x9cInhibition of the Arrest of Hematogenously Disseminated Tumor Cells,xe2x80x9d Cancer Metastases Rev. (1983) 2: 223-237. Tumor cells associated with such emboli then extravasate the circulation, invade and colonize target tissues. These newly established tumor colonies eventually grow and vascularize. Thrombin, however, is found tightly bound to fibrin and is also associated with the platelet surface via specific thrombin receptors. See Kumar et al, xe2x80x9cThe Influence of Fibrinogen and Fibrin on Thrombin Generation-Evidence for Feedback Activation of the Clotting System by Clot Bound Heparin,xe2x80x9d Thromb. Hemost. (1994) 72: 713-721; Liu et al, xe2x80x9cThe Binding of Thrombin to Fibrin,xe2x80x9d J. Biol. Chem. (1979) 254: 10421-10425; Smith at al., xe2x80x9cPlatelet Responses to Compound Interactions with Thrombinxe2x80x9d Biochemistry (1999) 38: 8936-8947; Kasirer-Friede et al, xe2x80x9cThrombin Receptor Occupancy Modulates Aggregation Efficiency and Platelet Surface Expression of vWF and Thrombospondin at Low Thrombin Concentrationsxe2x80x9d (1999) Thromb. Haemost. 81: 967-975. Thrombin is also a self antigen. Thus, whereas such emboli comprised of thrombin, fibrin and platelets are not readily susceptible to detection by the host immune system, the bound thrombin associated with the emboli is susceptible to detection by the activation of the natural serine protease inhibitor, heparin cofactor II, that is endogenous to the host. Heparin cofactor II has high selectivity for thrombin in its surface-associated or bound state. See, for example, Buchanan et al, xe2x80x9cEvidence for a Conformational Change of Surface-Bound Thrombin that Promotes Vessel Wall Thrombogenecity: Selective and Sustained Inhibition of its Activity by Intimatan but not by Heparin,xe2x80x9d Thromb. Haemost. (1999) ISTH Suppl.:413; Weitz et al, xe2x80x9cClot-Bound Thrombin Is Protected from Inhibition by Heparin-Antithrombin III but Is Susceptible to Inactivation by Antithrombin III-Independent Inhibitors,xe2x80x9d J. Clin. Invest. (1990) 86: 385-391; Buchanan et al, xe2x80x9cA Rationale for Targeting Antithrombotic Therapy at the Vessel Wall: Improved Antithrombotic Effect and Decreased Risk of Bleeding,xe2x80x9d Wien Klin Wochenschr (1999) 111: 81-89; Brister et al, xe2x80x9cEffect of Heparin and CL-0313 on Complement Activation In Vitro and Thrombin Generation During Cardiopulmonary Bypass In Vivo,xe2x80x9d Haemostasis (1996) 26: 575. Heparin cofactor II is ubiquitously distributed and is present in the systemic circulation and in the extravascular tissues. Thus, its biodistribution within the mammalian body is suitable to bind and neutralize thrombin in remote body residua potentially associated with occult disease states including infection and malignancies as well as the more accessible circulatory sites confined to the vascular compartments so associated with the venous and arterial thrombo-occlusive disorders.
Others have suggested using various glycosaminoglycans (GAGs) in combination with radioactive metal-ion chelates, alone or in combination with specific receptor-targeting peptides. For example, U.S. Pat. 5,561,201 (Dean et al), issued Oct. 1, 1996 and U.S. Pat. No. 5,714,579 (Dean et al), issued Feb. 3, 1998, disclose a radiolabeled imaging reagent composition comprised of a polybasic compound covalently linked to technetium-99 m binding moiety that is admixed with a polysulfated glycan, such as heparin, heparin sulfate, chondroitin sulfate or dermatan sulfate, and the composition being capable of binding to inflammation sites in vivo. See also U.S. Pat. No. 5,770,179 (Dean), issued Jun. 23, 1998, which discloses xcex2-glucans radiolabeled with technetium-99 m for diagnostic imaging where the imaging is mediated by the binding of the radiolabeled xcex2-glucan to xcex2-glucan receptors on monocytes, macrophages and neutrophils and thus relies on a cell-mediated inflammatory component associated with disease.
Another example is U.S. Pat. No. 5,707,606 (Ranney), issued Jan. 13, 1998, which discloses diagnostic agents comprised of a GAG, such as dermatan sulfate, essentially purified dermatan sulfate with a sulfur content of up to 9% and with selective oligosaccharide oversulfation, chondroitin sulfate, oversulfated chondroitin sulfate, and heparin sulfate, in non-covalent (ionic) and covalent combination with cationic, electrophilic metal-ion chelates that are alleged to improve the site delivery, uptake mechanism, sensitivity and kinetic-spatial profiles of the metal-ion chelate by directing the metal-ion chelate to sites of vascular inflammation.
There still remains a need for radiolabeled and radioactive diagnostic and therapeutic agents that are capable of more selectively targeting the site of potential disease or injury for diagnostic or treatment purposes, without the undesired side effects and problems of prior monoclonal antibody agents and synthetic peptides chelated to radioactive metals, and without the complications brought about by ionic formulations of chelates and targeting agents that promote the complex equilibria of self-associating systems in vivo. In particular, it would be desirable to provide radiolabeled and radioactive diagnostic and therapeutic agents and compositions that are capable of detecting vascular injury, disease, disorders and neovascularization processes, as well as treating such disease states, that are associated with the activation of thrombin and especially thrombin bound to disease sites.
The present invention relates to selectively targeted agents that comprise a dermatan sulfate having more than about 25% repeating L-iduronic acidxe2x86x924,6-di-O-sulfated N-acetyl-D-galactosamine disaccharide units that is covalently attached or bonded to a radioactive metal-ion binding moiety. The present invention also relates to the use of these targeted agents to prepare radiolabeled and/or radioactive diagnostic and/or therapeutic agents for diagnosing, and/or for treating disease states, disorders and the like. The present invention further relates to methods for diagnosing or treating such disease states which comprises administering a diagnostic and/or therapeutic amount of these selectively targeted radiolabeled and/or radioactive diagnostic and/or therapeutic agents to the person to be diagnosed or treated.
It has been surprisingly discovered that the targeted agents of the present invention are useful in preparing radiolabeled and/or radioactive diagnostic and/or therapeutic agents for the detection of diseased and/or rejuvenating endothelium associated with vascular injury, disease, disorders and/or neovascularization processes having a surface-bound thrombin component and, moreover, are superior and useful in therapeutic applications in the treatment of certain of such disease states having a surface-bound thrombin component. Of particular diagnostic utility are disease indications where the vascular pathology is associated with enhanced thrombogenicity, including but not limited to, thrombo-occlusive disorders involving clot formation, such as infarction, stroke, restenosis associated with percutaneous transluminal coronary angioplasty, coronary artery diseases such as atherosclerosis, peripheral vascular disease and cerebral vascular disease, as well as venous occlusive disorders such as deep vein thrombosis, and, a variety of malignancies involving hypercoagulopathies and vascularized tumor networks. In addition, the targeted radioactive metal ion binding moieties of the present invention are particularly useful in diagnostic and/or therapeutic applications involving the imaging and detection of occult malignancy, as well as the treatment and eradication thereof
The targeted agents of the present invention are preferential over prior monoclonal antibody- and peptide targeted agents having an attached metal ion chelate for diagnostic and therapeutic applications. In particular, the dermatan sulfate used in the present invention that is covalently bound or attached to the radioactive metal ion binding moiety is highly optimized in targeting surface-bound thrombin found in association with thrombi and thrombogenic vessels of the circulatory system, associated with a broad array of diverse disease states. The present invention is therefore broadly applicable to diagnostic and therapeutic applications pertaining to these diverse disease states and thus, is not limited by the complicated optimization and manufacturing issues associated with each unique peptide carrier/targeting agent. The targeted agents of the present invention are also advantageous in their lack of metabolism, their targeted biodistribution, and favorable pharmacokinetics and dynamics at diagnostically and therapeutically useful doses. Moreover, at such doses the anticoagulant effects of the targeted agents of the present invention are minimal when used in diagnostic and/or therapeutic applications, causing at most only negligible to mild elevations of activated clotting times (ACT). When used in association with severe disease states such as malignancy, mild ACT elevation is tolerable and moreover, considered beneficial, especially in association with thrombo-occlusive disorders where anticoagulation is a desired effect towards clinical outcome, even when used in diagnostic applications.
The targeted agents of the present invention also do not require cationic chelates or cationic peptides which mediate an association with a glycosaminoglycan (GAG) carrier by charge-charge attractive forces between the positively charged basic amines of the chelated metal ion moiety or peptide, and the negatively charged acidic functionalities of the GAG involving a principal mode of ionic binding. In particular, it has been found according to the present invention that: (1) cationic and electrophilic radioactive metal-ion chelates and cationic radiolabeled peptide-binding moieties are not required to achieve the selective and high-sensitivity detection of diseased tissues; and (2) ionic interactions between acidic mucopolysaccharide and a cationic metal ion chelate, or a cationic, peptide radiolabeled-binding moiety, can be avoided, thus also avoiding the complex issues of metabolism, biodistribution and safety considerations associated with self-associating systems comprising compositions of GAGs in ionic association with metal-binding agents, whether covalently or non-covalently bound to the targeting agent.